Microbiology

Collection of specimens and transport to the laboratory

General Guidelines

Collect specimens before commencement of antimicrobial therapy.  This is usually possible for most mild infections.  For more serious infections, antimicrobial therapy should not be withheld pending collection of a specific specimen.  For example, antimicrobial therapy should not be withheld pending collection of CSF from an individual with suspected meningitis or collection of sputum from an individual with severe pneumonia.  However, blood cultures can be obtained in nearly all cases prior to antimicrobial treatment of serious infection.

If in any doubt as to the appropriate container, please contact the laboratory for advice. 

Please send an adequate amount of specimen.  As a general rule – ‘the more specimen the better’. If pus is present, send pus rather than a swab and remember to send enough specimen if a whole series of tests are required.  This applies to CSF and serology specimens in particular.

Please ensure that relevant clinical details are included on the request form or on OCM (Order Communications – only available within St. James’s Hospital at present).  All of the above may influence the type of test that the laboratory performs.

Completion of Request Forms

Adequate identification of patient samples is essential for patient safety. The following details must be recorded on the request form:

  • Full Patient Name
  • DOB
  • Patients Address
  • External laboratory number/MRN (where available)
  • Gender
  • Date of Collection
  • Ward/Source
  • Requesting Clinician
  • Specimen Type
  • Tests requested

Those highlighted in bold are essential patient identifiers.

Specimens cannot be processed unless there is a minimum of two patient identifiers on the specimen which match those on the request form, one of which must be the full patient name.

Please ensure that relevant clinical details are included on the request form. For example, include details if the specimen is sent during an outbreak or there is a history of foreign travel or a specific diagnosis is being considered.  All of the above may influence the type of test that the laboratory performs.

Ordering by OCM.

Order communication is an electronic ordering system at present available only within the hospital.

  • Full name
  • Hospital number (MRN) if available
  • Date of birth
  • Ward / Location
  • Doctor in charge
  • Clinical details

When ordering by OCM, the above details will be included when the test is ordered and the information is on the barcode.

Specimen Identification

 The following details should be recorded on all specimens:

  • Full Patient Name
  • DOB
  • MRN (where available)
  • Date of collection

Those highlighted in bold are essential patient identifiers.

Specimens cannot be processed unless there is a minimum of two patient identifiers on the specimen which match those on the request form, one of which must be the full patient name. For those specimens where a higher level of patient confidentiality is required e.g. STI related specimens, patient initials, clinic number and DOB are required.

If the sample has been ordered by OCM this information will be on the OCM label.
The OCM label must be attached to the specimen in such a way that it does not cover the barcode on a Blood culture bottle, and is orientated to facilitate the reading of the barcode by the Laboratory computer system. OCM labels should not be wrapped around specimen containers as the barcode scanners cannot scan the information successfully.

It is laboratory policy NOT to process unlabelled or mis-labelled specimens.

CJD:  If a patient is at risk of developing a transmissible spongiform encephalopathy (TSE) or has a clinical syndrome compatible with a diagnosis of a TSE, tissue and CSF specimens must be labeled with the appropriate biohazard label as per CPL instructions. Other types of specimens may be sent without specified precautions. Please contact the medical staff in the laboratory prior to sample dispatch.

Swabs / containers available in SJH

  • Universal container – two types with different apertures are available for collection of bodily fluids – pus, urine, pleural fluid, CSF etc. but excluding blood.
  • Blue ‘Transwab’ for routine collection of specimens from skin, wounds etc.
  • Pernasal swab – for Bordetella pertussis. If required please contact the Medical Staff in the Microbiology Department.
  • Abbott multi-Collect Specimen Collection Kit TM to collect urine specimens or to collect swab specimens from the male urethra, the endocervix, the vagina, the pharynx and/or the rectum for chlamydia / Neisseria gonorrhoeae diagnosis.
  • 20 ml Red topped bottle for serum. Send 5 -10 ml of a clotted specimen of blood.
  • FBC bottle with EDTA as an anticoagulant for plasma – send 5 ml of blood.
  • New York City Medium is available for culture of Neisseria gonorrhoeae (N.B Store at 4ºC and return to room temperature before use.)
  • Blood culture bottles:
    • Aerobic bottle
    • Anaerobic bottle
    • Mycobacterial bottle Bactec MYCO/F LYTIC
  • Viral transport medium and viral swabs: These are available on request. Please consult the Laboratory Diagnosis of Viral infections handbook for further details, or contact the Microbiology Medical Staff.

Transport of Specimens to the laboratory

Specimens should be transported to the laboratory without delay to ensure optimal results.

All specimen containers must be tightly closed and placed in a transparent hazard bag for transport to the laboratory.
It is the responsibility of the person dispatching the specimen to the laboratory to ensure that it is packaged correctly, and does not pose a risk to anyone coming in contact with it during transport or on receipt in the laboratory.

All CSF specimens are treated with priority in the Microbiology Laboratory. Outside normal hours the requesting clinician must ensure that the on call medical scientist in Microbiology is aware that a CSF is expected.

Sample retention in the Laboratory and further testing requests

Samples are generally retained in the Microbiology Laboratory for 72hrs. Requests for further testing on samples may be possible on a case-by-case basis. Please contact the Laboratory as soon as possible if the need for further testing is identified.

Recommended samples to be sent from patients with particular clinical syndromes

Suspected bacteraemia, Systemic Inflammatory Response Syndrome (SIRS), Sepsis, Septic Shock

Blood cultures - For optimum sensitivity, two sets of blood cultures should be collected from separate sites within a 24 h period. These should be taken at least 20 min apart.  For patients with suspected endocarditis, three sets should be collected.

Method:  Observe standard precautions, wash hands, carefully disinfect the skin with alcohol, allow to dry, wear sterile gloves, insert vacutainer into vein, collect 10 ml of blood into an aerobic and 10 ml into an anaerobic blood culture bottle.  Please see Infection Control Manual for detailed description of blood culture technique. Yeasts and fungi may be detected in the normal blood culture system.

Look for a focus of infection and culture those sites appropriate to a suspected focus.

CNS infections

Blood cultures

Blood cultures should be collected from all patients with suspected meningitis.

CSF

CSF should be collected from all adult patients with suspected meningitis except when a clear contraindication exists (e.g. signs of raised intra-cranial pressure, focal neurological signs, severe shock, severely depressed or fluctuating conscious level, coagulation disorder) or if there is a confident clinical diagnosis of meningococcal infection with a typical rash. Note antimicrobials should NOT be withheld pending a lumbar puncture.

Plasma

Send 5 ml EDTA blood for meningococcal or Pneumococcal PCR.  See virology manual for viral diagnosis.

Paired sera

Paired sera (two specimens taken 10-14 days apart) may be useful for a retrospective diagnosis of meningococcal disease.

Respiratory tract infection

Tonsillopharyngitis
Send a throat swab.  Please contact the laboratory if diphtheria or pertussis is suspected.

Sinusitis:
Using a syringe aspiration technique, a specially trained physician or an ENT surgeon can obtain material from maxillary, frontal, or other sinuses.   Place the contents of the syringe into a sterile universal container.

Otitis media:
Usually no specimens are forwarded to the laboratory.

Diagnosis of Whooping cough:
Please discuss with laboratory medical staff.

Bronchitis:
A good quality purulent or mucopurulent sputum specimen should be obtained, preferably before antimicrobial therapy.

Pneumonia:
It is not necessary to perform a full range of microbiological investigations on all patients with community-acquired pneumonia. The extent of investigation should be determined by the severity and clinical course.  Specimens that should/may be sent include:

Blood cultures should be obtained from all patients.

Sputum: A good quality purulent or mucopurulent sputum specimen should be obtained, preferably before antimicrobial therapy although antimicrobial therapy should not be delayed unnecessarily while awaiting a sputum specimen.  The specimen should be transported to the laboratory within 2 h.  Salivary or mucosalivary specimens are unsuitable and as such are not processed.

Urine for Legionella antigen should be obtained from all patients with severe CAP and particular patients with specific risk factors.

Paired sera should be obtained for all patients who do not respond to β-lactam antibiotics and particular patients with specific risk factors.

Pleural fluid: If a pleural effusion is present, consider aspiration into a sterile universal container at an early stage.

Bronchoscopic samples may also be required, especially among immunocompromised patients.

Pneumocystis jiroveci: diagnosis of Pneumocystis is carried out on bronchoscopic samples in cytology.

Gastrointestinal tract infection

Gastroenteritis
Please note that this laboratory employs a cost-effective approach to the diagnosis of infectious diarrhoea.  Not all specimens are examined for every pathogen.  It is therefore important that clinical details or suspected diagnoses are included on the request form or OCM. Information that is of use when processing specimens includes: travel history, relationship to a particular food, prolonged diarrhoea, antibiotic use, suspected outbreak.  The laboratory examines all stool samples routinely for:
Salmonella
Shigella
E. coli O157
Campylobacter.
Clostridium difficile toxin detection is performed on all specimens from in-patients. 
Other pathogens e.g. Cryptosporidium, Yersinia, Vibrio, Aeromonas, ova and parasites etc. are only examined if the clinical details suggest that possibility. 

Please note the possibility of Norovirus infection and state whether vomiting is a feature or whether an outbreak is suspected.  Please send a blood culture if typhoid fever is suspected.

When to send a stool specimen: Send a stool specimen to the laboratory when there are ≥3 liquid or very loose stools per day.  There may be other symptoms suggestive of infectious diarrhoea e.g. abdominal pain or discomfort, nausea, faecal urgency, tenesmus, fever, blood or mucus in stools. Within the hospital specimens must be sent to the laboratory immediately. In General Practice, please refrigerate if there is to be a delay in transporting the specimen.
How many samples to send: One stool specimen is normally all that is required for culture. As microscopy for parasites is less sensitive, please send 3 specimens (but no more than 3) on different days as some parasites are excreted intermittently.  If a worm is excreted, please send the worm and faeces sample.

How much to send:  Please fill the specimen container to between ¼ and ½ full. Please do not fill to the brim. 

Upper gastro-intestinal specimens for culture of H. pylori
Please contact the laboratory prior to sending. Obtain a corpus and antral biopsy.  Place in a universal container and transport to the laboratory as soon as possible - certainly within 3 hours.

Anal swabs
Anal swabs are collected primarily for the detection of carriage of Vancomycin-Resistant Enterococci (VRE) or for culture for Neisseria gonorrhoeae.  Insert swab about 2 cm into anal cavity, rotate and send specimen to laboratory.

Bone and joint infection.

Osteomyelitis

Blood culture:  Blood cultures should be performed an all patients with suspected osteomyelitis, preferably before antibiotics are started. 

Bone biopsy: A biopsy of bone is the preferred specimen for the establishment of a diagnosis of osteomyelitis and the causative agent. The biopsy should be placed in a sterile universal container with saline and transported to the laboratory as quickly as possible.  It is also preferable to send multiple specimens (3 or 4) especially in cases of infection associated with a prosthetic device as this makes interpretation easier if a skin organism is recovered.  Consider requesting mycobacterial culture from high-risk groups.

Septic arthritis

Blood cultures: Blood cultures should be performed an all patients with septic arthritis, preferably before antibiotics are started.

Joint aspirate: A joint aspirate obtained using an aseptic technique should be submitted in a sterile universal container from all patients with septic arthritis.

Polyarticular arthritis

Send blood cultures and joint aspirate.  Consider sending serum for Lyme disease antibodies.  Viral causes also include Parvovirus and Rubella.  See Virology user’s manual for appropriate specimens to take.

Chronic septic arthritis

Consider requesting serum for antibodies to Brucella, culture of joint aspirate for mycobacteria and fungi.

Reactive arthritis

Faeces culture may be requested for Salmonella, Shigella, Campylobacter and Yersinia.

Send a serum specimen and request antibodies to Campylobacter and Yersina. In rheumatic fever, send a throat swab and serum for ASO titre.

If a sexually transmitted aetiology is suspected then urethral, cervical or rectal swabs may be taken for gonococcal or chlamydial detection.

Urine specimens

When should you send a sample of urine:

It is probably reasonable to treat a young sexually active female with symptoms of simple cystitis empirically but a urine specimen should be sent for microbiological examination from all other cases.  In severe or complicated UTI, a follow-up specimen should be taken 5 days post completion of antibiotic therapy.  Persistence of bacteriuria implies a structural abnormality. 

A specimen should be sent from patients with symptoms as asymptomatic bacteriuria is generally not a cause for concern except in pregnant women and patients undergoing surgery on the g-u tract. The role of asymptomatic bacteriuria in children is controversial.

The same applies to patients with in-dwelling urinary catheters.  Bacteriuria occurs in the vast majority of patients who are catheterised for more than 5 days, a urine specimen should only be sent if there are symptoms or signs suggestive of a urine or a systemic infection.

What type of specimen should you send? 

Send a mid-stream specimen of urine (MSU) where possible.  Patients should be instructed to pass a little urine into the toilet first, then pass enough urine into the specimen container to half fill it and finish urinating into the toilet.  Never obtain urine from a bedpan or commode.  Obtain about 10 ml of urine in a sterile universal container tighten the lid and transport to the laboratory without delay.  Specimens should be processed within 4 h.  In General Practice if transport to the laboratory has to be delayed, the specimen can be stored at 4°C for up to 48 h. 

A clean catch urine may also be obtained if the patient cannot co-operate.

A catheter specimen of urine (CSU) may also be sent to the laboratory.  Urine should be obtained from an already catheterised patient by a syringe and needle from the catheter before it enters the collection bag.  Clean the access point with a swab saturated with 70% isopropyl alcohol and allow time to dry. Using a sterile syringe and needle (if necessary), aspirate the required amount of urine from the access point.  Re-clean access point with a swab saturated with 70% isopropyl alcohol.

Genital tract specimens

Vulva / penile lesions

Syphilis - Please contact the laboratory if diagnosis based on dark-ground microscopy is appropriate.  Clean the surface of any lesion with sterile normal saline. If there is a crust, gently remove it. Abrade the lesion until serous fluid (not blood) emerges. Wipe away fluid and debris with sterile gauze. Press the base of lesion until clear fluid is expressed.  Aspirate some of the discharge into a small plastic pipette (obtainable from Microbiology Lab) and place pipette into a sterile universal container. Tighten lid and send for dark-field microscopy immediately.

Molluscum contagiosum and condyloma acuminata - punch biopsy for histological examination if necessary.

Bartholins gland Disinfect the skin with povidine iodine. In the early stage of bartholinitis, try to express secretions from the openings of the glands. Collect the exudates with a swab or a syringe. With the formation of abscess, aspirate material from the gland with a syringe.

Vaginal specimens

Obtain a high vaginal swab by use of a speculum and a trans swab and submit to the laboratory.

Cervical / endocervical swabs:  Use a speculum without lubricant. Wipe the cervix clean of vaginal secretions and mucus. Gently insert a swab into the endocervical canal and rotate to obtain any exudate.

Chlamydia trachomatis /Neisseria gonorrhoeae:  To detect C. trachomatis / Neisseria gonorrhoeae, the laboratory currently uses the Abbott RealTime CT / NG System.

Sample Collection and Transport

The Abbott multi-Collect Specimen Collection Kit should be used to collect urine specimens or to collect swab specimens from the male urethra, the endocervix, the vagina, the pharynx and/or the rectum only.

Use only the orange shaft swab provided in the Abbott multi-Collect Specimen Collection Kit for collecting male urethral, endocervical, or vaginal specimens.

The swab must remain in the Transport Tube after specimen collection. Do not place multiple swabs or a combination of swab and urine in the Transport Tube

Add urine to the Transport Tube until the liquid level falls within the fill window on the tube label.

The presence of blood, mucus, some spermicidal agents, feminine powder sprays, and treatments for vaginal conditions such as yeast infection may interfere with nucleic acid test (NAT) based assays. The effects of other factors such as vaginal discharge, use of tampons, douching, or
specimen collection variables have not been determined.

Urine Specimen Collection:

  • The patient should not have urinated for at least one hour prior to sample collection.
  • Discard specimen collection swab; it is not required for urine specimen collection.
  • Using a urine specimen collection cup, the patient should collect the first 20 to 30 mL of voided urine (the first part of the stream).
  • Unscrew the transport tube cap, taking care not to spill the transport buffer within.
  • Handle the cap and tube carefully to avoid contamination.
  • Use the plastic transfer pipette to transfer urine from the collection cup into the transport tube until the liquid level in the tube falls within the clear fill window of the transport tube label or a new specimen should be collected. Do not overfill. Slightly more than one full squeeze of the transfer pipette bulb may be required to transfer the necessary volume of urine specimen.
  • Recap the transport tube carefully. Ensure the cap seals tightly
  • Label the transport tube with sample identification information, including date of collection. Take care not to obscure the fill window on the tube label.
  • Decontaminate and dispose of all specimens, reagents, and other potentially contaminated materials in accordance with local regulations.

Endocervical Swab Specimen Collection:

  • Discard disposable transfer pipette; it is not required for endocervical swab specimen collection.
  • Remove the sterile swab from the wrapper.
  • Insert the white tip of the specimen collection swab into the endocervical canal.
  • Gently rotate the swab for 15 to 30 seconds to ensure adequate sampling.
  • Withdraw the swab carefully.
  • Handle the cap and tube carefully to avoid contamination.
  • Unscrew the transport tube cap and immediately place the specimen collection swab into the transport tube so that the white tip is down.
  • Carefully break the swab at the scored line on the shaft; use care to avoid splashing of contents
  • Recap the transport tube. Ensure the cap seals tightly.
  • Label the transport tube with sample identification information, including date of collection.

Vaginal Swab Specimen Collection

  • Discard disposable transfer pipette; it is not required for vaginal swab specimen collection.
  • Remove the sterile swab from the wrapper.
  • Insert the white tip of the specimen collection swab about two inches (5 cm) into the opening of the vagina.
  • Gently rotate the swab for 15 to 30 seconds against the sides of the vagina.
  • Withdraw the swab carefully.
  • Handle the cap and tube carefully to avoid contamination.
  • Unscrew the transport tube cap and immediately place the specimen collection swab into the transport tube so that the white tip is down.
  • Carefully break the swab at the scored line on the shaft; use care to avoid splashing of contents.
  • Recap the transport tube. Ensure the cap seals tightly.

Label the transport tube with sample identification information, including date of collection.

Male Urethral Swab Specimen Collection

  • The patient should not have urinated for at least one hour prior to sample collection.
  • Discard disposable transfer pipette; it is not required for male urethral swab specimen collection.
  • Remove the sterile swab from the wrapper.
  • Insert the white tip of the specimen collection swab 3/4 to 11/2 inches (2 to 4 cm) into the urethra.
  • Gently rotate the swab for 2 to 3 seconds to ensure adequate sampling.
  • Withdraw the swab carefully.
  • Handle the cap and tube carefully to avoid contamination.
  • Unscrew the transport tube cap and immediately place the specimen collection swab into the transport tube so that the white tip is down.
  • Carefully break the swab at the scored line on the shaft; use care to avoid splashing of contents.
  • Recap the transport tube. Ensure the cap seals tightly.
  • Label the transport tube with sample identification information, including date of collection.

Rectal Swab Specimen Collection

Used to detect rectal carriage and infection due to Chlamydia / N. gonorrhoeae and sometimes Herpes simplex virus. Pass the tip of a sterile swab approximately 2.5 cm beyond the anal sphincter. Rotate the swab gently and withdraw it into the appropriate transport medium.

Collect endometrial specimens by transcervical aspiration through a telescoping catheter.

Endometrial specimen collection

Collect endometrial specimens by transcervical aspiration through a telescoping catheter.

Chlamydia/Gonorrhoea specimen transport to the Laboratory and Specimen storage

After collection, transport and store transport tube at 2°C to 30°C for up to 14 days. If longer storage is needed, store at -10°C or colder for up to 90 days. Specimens should be packaged and labelled in compliance with applicable regulations covering the transport of clinical, diagnostic, or biological specimens.

Time and temperature conditions for storage of specimens for Chlamydia / Gonorrhoea must be adhered to during transport.

NB. If Urethral / rectal specimens for the detection of Neisseria gonorrhoeae are inoculated directly onto New York City medium, ensure the medium is within the marked expiry date. The plates should be stored at 4º C and returned to room temperature before inoculating.
Transport the inoculated plates to the Laboratory as soon as possible. Recovery of organisms may be affected by inappropriate storage of New York City medium

Skin and superficial wound swabs

Note that routine sampling of skin lesions that do not appear clinically infected should generally not be performed.  If there is a clinically infected lesion, please send a sample of pus in a universal container wherever possible.  Pus is always preferable to a swab.  If there is insufficient specimen, then use a ‘Transwab’, sample the infected area and send to the laboratory.

Deep-seated wounds/abscesses/ post-operative wound infection

Please send a sample of pus in a universal container wherever possible. Pus is always preferable to a swab.  If there is insufficient specimen, then use a ‘Transwab’, sample the infected area and send to the laboratory. Clean the surface of the wound with sterile saline or water before taking the swab.

Mycobacterial Infection

The diagnosis of mycobacterial infection requires special staining and culture techniques.  In the future, the routine diagnosistic service will be undertaken by the Irish Mycobacterial Reference Laboratory (IMRL), located at St. James’s Hospital.  Please ensure that you request TB culture on the request form or OCM if the diagnosis is suspected.

Suitable specimens:
The following is a list of suitable specimens to submit:

  • Good quality early morning Sputum
  • Specimens obtained at Bronchoscopy
  • Pus
  • CSF, Pleural, Peritoneal, Joint and other Sterile Fluids
  • Tissue
  • Lymph node biopsy
  • Pus aspirated from lymph nodes
  • Pleural biopsy
  • Surgical sample for routine culture
  • Radiological sample for routine culture
  • Histology sample where non-respiratory TB is a possibility
  • Aspiration sample where non-respiratoryTB is a possibility
  • Bone
  • Gastric aspiration
  • Blood
  • Bone marrow
  • Urine in certain circumstances

Unsuitable specimens:
The following is a list of unsuitable specimens that will usually be rejected by the laboratory:

  • Poor quality sputum specimens e.g. salivary specimens or specimens of minute quantities
  • Faeces
  • Urine, except when: A diagnosis of renal tuberculosis is suspected or the patient is immunocompromised or following prior discussion with a laboratory doctor.

An early morning MSU or CSU sample, taken into a sterile plastic container, should be procured and immediately submitted to the IMRL on each of three consecutive days.

Sputum specimens:  Three consecutive early morning specimens should be submitted before the commencement of therapy.  The specimen should be coughed from deep within the lungs.

Specimens obtained at bronchoscopy:  Specimens should be placed in a sterile universal container and transported to the laboratory without delay.

Tissue:  Tissue is preferable to necrotic material.  Do not place any fixatives in the sterile universal container.  If there is a possibility that the specimen may dry out before it reaches the laboratory, then sterile saline may be added to the container

Blood:  Blood may be useful for the diagnosis of non-tuberculous mycobacterial infection in profoundly immunosuppressed individuals. Please contact the laboratory to obtain a Bactec MYCO/F LYTIC bottle.  Add 7-10 ml of blood to the Bactec MYCO/F LYTIC bottle and submit to the laboratory.

Bone marrow:  The volume of bone marrow obtained determines how the specimen should be collected.  Specimens of less than 0.5 ml should be taken into a plastic sterile universal container.  Specimens of greater than 0.5 ml should be inoculated directly into a Bactec MYCO/F LYTIC blood culture bottle.  Please contact the laboratory to obtain a Bactec MYCO/F LYTIC bottle. 

CSF/sterile bodily fluids:  The yield from examination of CSF specimens is dependant on the volume obtained.  Ideally 10 ml should be obtained.  Similarly, about 10 ml should be submitted for mycobacterial culture from other normally sterile bodily fluids e.g. pleural, ascitic, joint.

Fungal nail and skin infections

Affected areas should be scraped with a blunt scalpel to harvest affected hairs, broken-off hair stubs and scalp scale. This is preferable to plucking, which may remove uninvolved hairs. Scrapings should be transported in a folded square of paper preferably fastened with a paper clip, but commercial packs are also available (e.g. ‘Mycotrans’). It is easier to see affected hairs on white paper rather than black.